Satoru Shinkuma

Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1α in autosomal recessive hypotrichosis†

Autosomal recessive hypotrichosis (ARH) is characterized by sparse hair on the scalp without other abnormalities. Three genes, DSG4, LIPH, and LPAR6 (P2RY5), have been reported to underlie ARH. We performed a mutation search for the three candidate genes in five independent Japanese ARH families and identified two LIPH mutations: c.736T>A (p.Cys246Ser) in all five families, and c.742C>A (p.His248Asn) in four of the five families. Out of 200 unrelated control alleles, we detected c.736T>A in three alleles and c.742C>A in one allele. Haplotype analysis revealed each of the two mutant alleles is derived from a respective founder. These results suggest the LIPH mutations are prevalent founder mutations for ARH in the Japanese population. LIPH encodes PA‐PLA1α (LIPH), a membrane‐associated phosphatidic acid‐preferring phospholipase A1α. Two residues, altered by these mutations, are conserved among PA‐PLA1α of diverse species. Cys246 forms intramolecular disulfide bonds on the lid domain, a crucial structure for substrate recognition, and His248 is one amino acid of the catalytic triad. Both p.Cys246Ser‐ and p.His248Asn‐PA‐PLA1α mutants showed complete abolition of hydrolytic activity and had no P2Y5 activation ability. These results suggest defective activation of P2Y5 due to reduced 2‐acyl lysophosphatidic acid production by the mutant PA‐PLA1α is involved in the pathogenesis of ARH. Hum Mutat 31:1–9, 2010.

An annexin A1–FPR1 interaction contributes to necroptosis of keratinocytes in severe cutaneous adverse drug reactions

Annexin A1 secreted from drug-stimulated monocytes contributes to keratinocyte necroptosis in serious drug-related adverse events in skin. Subduing a Severe Skin Side Effect Certain pain relievers and antiepileptic drugs can cause a very rare, but sometimes fatal, side effect in which skin painfully blisters and peels, caused by the patients’ immune response to the drug. Saito et al. now find that, in susceptible patients, the drug causes secretion of the protein annexin A1 from immune cells, with deadly effect on skin cells. Annexin acts on these cells to cause necroptosis, a programmed form of cell death. The authors confirmed their results in mice, showing that an inhibitor of necroptosis blocked skin blistering. With these findings, Saito et al. lay the groundwork for a countermeasure to this dangerous side effect of otherwise extremely beneficial drugs. Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening, cutaneous adverse drug reactions that are accompanied by keratinocyte cell death. Dead keratinocytes from SJS/TEN lesions exhibited necrosis, by morphological criteria. Supernatant from peripheral blood mononuclear cells (PBMCs) that had been exposed to the causative drug from patients with SJS/TEN induced the death of SJS/TEN keratinocytes, whereas supernatant from PBMCs of patients with ordinary drug skin reactions (ODSRs) exposed to the same drug did not. Keratinocytes from ODSR patients or from healthy controls were unaffected by supernatant from SJS/TEN or ODSR PBMCs. Mass spectrometric analysis identified annexin A1 as a key mediator of keratinocyte death; depletion of annexin A1 by a specific antibody diminished supernatant cytotoxicity. The necroptosis-mediating complex of RIP1 and RIP3 was indispensable for SJS/TEN supernatant–induced keratinocyte death, and SJS/TEN keratinocytes expressed abundant formyl peptide receptor 1 (FPR1), the receptor for annexin A1, whereas control keratinocytes did not. Inhibition of necroptosis completely prevented SJS/TEN-like responses in a mouse model of SJS/TEN. Our results demonstrate that a necroptosis pathway, likely mediated by annexin 1 acting through the FPR1 receptor, contributes to SJS/TEN.

Antibodies to Pathogenic Epitopes on Type XVII Collagen Cause Skin Fragility in a Complement-Dependent and -Independent Manner

In bullous pemphigoid (BP), the most prevalent autoimmune blistering disease, type XVII collagen (COL17) is targeted by circulating autoantibodies. BP is thought to be an autoantibody-mediated complement-fixing blistering disease, and a juxtamembranous noncollagenous 16A (NC16A) domain spanning Glu490 to Arg566 was proved to be the main pathogenic region on COL17, although precise pathogenic epitopes within NC16A have not been elucidated. In this study, we showed that injection of rabbit IgG Abs targeting Asp522 to Gln545 induced skin fragility associated with in vivo deposition of IgG and complement in neonatal COL17-humanized mice. Notably, immunoadsorption of rabbit anti-NC16A IgG Ab with this epitope (Asp522 to Gln545) or the anti-NC16A IgG administered together with the peptides of this epitope as a decoy ameliorated skin fragility in the injected neonatal COL17-humanized mice compared with the anti-NC16A IgG alone even though all of the mice showed both IgG and complement deposition. These results led us to investigate an additional, complement-independent mechanism of skin fragility in the mice injected with anti-COL17 Abs. The rabbit anti-NC16A IgG depleted the expression of COL17 in cultured normal human keratinocytes, whereas immunoadsorption of the same IgG with this epitope significantly suppressed the depletion effect. Moreover, passive transfer of F(ab′)2 fragments of the human BP or rabbit IgG Abs against COL17 demonstrated skin fragility in neonatal COL17-humanized mice. In summary, this study reveals the importance of Abs directed against distinct epitopes on COL17, which induce skin fragility in complement-dependent as well as complement-independent ways.

Bullous Pemphigoid Autoantibodies Directly Induce Blister Formation without Complement Activation

Complement activation and subsequent recruitment of inflammatory cells at the dermal/epidermal junction are thought to be essential for blister formation in bullous pemphigoid (BP), an autoimmune blistering disease induced by autoantibodies against type XVII collagen (COL17); however, this theory does not fully explain the pathological features of BP. Recently, the involvement of complement-independent pathways has been proposed. To directly address the question of the necessity of the complement activation in blister formation, we generated C3-deficient COL17-humanized mice. First, we show that passive transfer of autoantibodies from BP patients induced blister formation in neonatal C3-deficient COL17-humanized mice without complement activation. By using newly generated human and murine mAbs against the pathogenic noncollagenous 16A domain of COL17 with high (human IgG1, murine IgG2), low (murine IgG1), or no (human IgG4) complement activation abilities, we demonstrate that the deposition of Abs, and not complements, is relevant to the induction of blister formation in neonatal and adult mice. Notably, passive transfer of BP autoantibodies reduced the amount of COL17 in lesional mice skin, as observed in cultured normal human keratinocytes treated with the same Abs. Moreover, the COL17 depletion was associated with a ubiquitin/proteasome pathway. In conclusion, the COL17 depletion induced by BP autoantibodies, and not complement activation, is essential for the blister formation under our experimental system.

Human IgG1 Monoclonal Antibody against Human Collagen 17 Noncollagenous 16A Domain Induces Blisters via Complement Activation in Experimental Bullous Pemphigoid Model

Bullous pemphigoid (BP) is an autoimmune blistering disease caused by IgG autoantibodies targeting the noncollagenous 16A (NC16A) domain of human collagen 17 (hCOL17), which triggers blister formation via complement activation. Previous in vitro analysis demonstrated that IgG1 autoantibodies showed much stronger pathogenic activity than IgG4 autoantibodies; however, the exact pathogenic role of IgG1 autoantibodies has not been fully demonstrated in vivo. We constructed a recombinant IgG1 mAb against hCOL17 NC16A from BP patients. In COL17-humanized mice, this mAb effectively reproduced a BP phenotype that included subepidermal blisters, deposition of IgG1, C1q and C3, neutrophil infiltration, and mast cell degranulation. Subsequently, alanine substitutions at various C1q binding sites were separately introduced to the Fc region of the IgG1 mAb. Among these mutated mAbs, the one that was mutated at the P331 residue completely failed to activate the complement in vitro and drastically lost pathogenic activity in COL17-humanized mice. These findings indicate that P331 is a key residue required for complement activation and that IgG1-dependent complement activation is essential for blister formation in BP. This study is, to our knowledge, the first direct evidence that IgG1 Abs to hCOL17 NC16A can induce blister formation in vivo, and it raises the possibility that IgG1 mAbs with Fc modification may be used to block pathogenic epitopes in autoimmune diseases.

Plectin expression patterns determine two distinct subtypes of epidermolysis bullosa simplex

Plectin is a cytoskeletal linker protein that has a dumbbell‐like structure with a long central rod and N‐ and C‐terminal globular domains. Mutations in the gene encoding plectin (PLEC1) cause two distinct autosomal recessive subtypes of epidermolysis bullosa (EB): EB simplex with muscular dystrophy (EBS‐MD), and EB simplex with pyloric atresia (EBS‐PA). Here, we demonstrate that normal human fibroblasts express two different plectin isoforms including full‐length and rodless forms of plectin. We performed detailed analysis of plectin expression patterns in six EBS‐MD and three EBS‐PA patients. In EBS‐PA, expression of all plectin domains was found to be markedly attenuated or completely lost; in EBS‐MD, the expression of the N‐ and C‐terminal domains of plectin remained detectable, although the expression of rod domains was absent or markedly reduced. Our data suggest that loss of the full‐length plectin isoform with residual expression of the rodless plectin isoform leads to EBS‐MD, and that complete loss or marked attenuation of full‐length and rodless plectin expression underlies the more severe EBS‐PA phenotype. These results also clearly account for the majority of EBS‐MD PLEC1 mutation restriction within the large exon 31 that encodes the plectin rod domain, whereas EBS‐PA PLEC1 mutations are generally outside exon 31. Hum Mutat 30:1–9, 2010.

A Novel Humanized Neonatal Autoimmune Blistering Skin Disease Model Induced by Maternally Transferred Antibodies

All mammal neonates receive maternal Abs for protection against pathogenic organisms in the postnatal environment. However, neonates can experience serious adverse reactions if the Abs transferred from the mother recognize self-molecules as autoAgs. In this study, we describe a novel model for autoimmune disease induced by transferred maternal Abs in genetically transformed Ag-humanized mice progeny. Bullous pemphigoid is the most common life-threatening autoimmune blistering skin disease that affects the elderly, in which circulating IgG autoAbs are directed against epidermal type XVII collagen (COL17). We have established a genetically manipulated experimental mouse model in which maternal Abs against human COL17 are transferred to pups whose skin expresses only human and not mouse COL17, resulting in blistering similar to that seen in patients with bullous pemphigoid. Maternal transfer of pathogenic Abs to humanized neonatal mice is a unique and potential experimental system to establish a novel autoimmune disease model.

Plectin Deficiency Leads to Both Muscular Dystrophy and Pyloric Atresia in Epidermolysis Bullosa Simplex

Plectin is a cytoskeletal linker protein which has a long central rod and N‐ and C‐terminal globular domains. Mutations in the gene encoding plectin (PLEC) cause two distinct autosomal recessive subtypes of epidermolysis bullosa: EB simplex (EBS) with muscular dystrophy (EBS‐MD), and EBS with pyloric atresia (EBS‐PA). Previous studies have demonstrated that loss of full‐length plectin with residual expression of the rodless isoform leads to EBS‐MD, whereas complete loss or marked attenuation of expression of full‐length and rodless plectin underlies the more severe EBS‐PA phenotype. However, muscular dystrophy has never been identified in EBS‐PA, not even in the severe form of the disease. Here, we report the first case of EBS associated with both pyloric atresia and muscular dystrophy. Both of the premature termination codon‐causing mutations of the proband are located within exon 32, the last exon of PLEC. Immunofluorescence and immunoblot analysis of skin samples and cultured fibroblasts from the proband revealed truncated plectin protein expression in low amounts. This study demonstrates that plectin deficiency can indeed lead to both muscular dystrophy and pyloric atresia in an individual EBS patient.

Ultrastructure and molecular pathogenesis of epidermolysis bullosa

Epidermolysis bullosa (EB) is classified into the three major subtypes depending on the level of skin cleavage within the epidermal keratinocyte or basement membrane zone. Tissue separation occurs within the intraepidermal cytoplasm of the basal keratinocyte, through the lamina lucida, or in sublamina densa regions of the basal lamina (basement membrane) in EB simplex, junctional EB, and dystrophic EB, respectively. Transmission electron microscopy (TEM) is an effective method for determining the level of tissue separation and hemidesmosome (HD) and anchoring fibril morphology if performed by experienced operators, and has proven to be a powerful technique for the diagnosis of new EB patients. Recent advances in genetic and immunofluorescence studies have enabled us to diagnose EB more easily and with greater accuracy. This contribution reviews TEM findings in the EB subtypes and discusses the importance of observations in the molecular morphology of HD and basement membrane associated structures.

Keratinocyte-/Fibroblast-Targeted Rescue of Col7a1-Disrupted Mice and Generation of an Exact Dystrophic Epidermolysis Bullosa Model Using a Human COL7A1 Mutation

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe hereditary bullous disease caused by mutations in COL7A1, which encodes type VII collagen (COL7). Col7a1 knockout mice (COL7(m-/-)) exhibit a severe RDEB phenotype and die within a few days after birth. Toward developing novel approaches for treating patients with RDEB, we attempted to rescue COL7(m-/-) mice by introducing human COL7A1 cDNA. We first generated transgenic mice that express human COL7A1 cDNA specifically in either epidermal keratinocytes or dermal fibroblasts. We then performed transgenic rescue experiments by crossing these transgenic mice with COL7(m+/-) heterozygous mice. Surprisingly, human COL7 expressed by keratinocytes or by fibroblasts was able to rescue all of the abnormal phenotypic manifestations of the COL7(m-/-) mice, indicating that fibroblasts as well as keratinocytes are potential targets for RDEB gene therapy. Furthermore, we generated transgenic mice with a premature termination codon expressing truncated COL7 protein and performed the same rescue experiments. Notably, the COL7(m-/-) mice rescued with the human COL7A1 allele were able to survive despite demonstrating clinical manifestations very similar to those of human RDEB, indicating that we were able to generate surviving animal models of RDEB with a mutated human COL7A1 gene. This model has great potential for future research into the pathomechanisms of dystrophic epidermolysis bullosa and the development of gene therapies for patients with dystrophic epidermolysis bullosa.